“Serum TSH and Papillary Thyroid Cancer”
PhD Program DirectorProf. Stefano Del Prato
Tutor:
Prof. Paolo Vitti
PhD Candidate Maria Annateresa Provenzale
1 INDEX SUMMARY pg 3 INTRODUCTION pg 5 Thyroid nodules pg 5 Prevalence pg 5 Clinical Presentation pg 5 Ultrasound Pattern pg 6 Fine needle aspiration pg 8 Cytological classification pg 9 Thyroid cancer pg 10 Incidence pg 10 Risk Factors pg 11 Histological classification pg 11 Molecular features pg 12 RET/PTC pg 12 B-RAF pg 13 RAS pg 13 Papillary microcarcinoma pg 14 Definition and prevalence pg 14
Clinical presentation pg 14 Molecular features pg 15
Clinical impact and therapy pg 15 TSH role pg 16 TSH action pg 16 Clinical relevance pg 17
TSH and microcarcinoma pg 18
STUDY pg 20 Aim of the study pg 20 Patients and methods pg 20
2 Study design pg 20 Patients pg 20 Inclusion criteria pg 21 Exclusion criteria pg 21 Diagnostic procedures pg 21 Indication for thyroidectomy pg 22 Thyroid function tests pg 23 Fine needle aspiration and cytology pg 23 Histological examination pg 23 Statistic Analysis pg 24 Results pg 25 Study group pg 25 Tumor size and limph node metastasis pg 26 Histological variants pg 26 Multifocality pg 27 TSH pg 27 Discussion pg 29 Conclusions pg 33 References pg 34 Figure Legend pg 44 Tables and figures
3 SUMMARY
Papillary carcinoma is the most common thyroid cancer and its incidence has been increasing in recent years, largely due to increased detection of small lesions. TSH is an important growth factor for thyroid follicular cells. In literature it is well known that there is a relationship between serum TSH and papillary thyroid carcinoma (PTC): higher TSH levels are associated with a higher frequency of papillary carcinoma (even when serum TSH is in the normal range) and with a more advanced cancer stage. Aim of this project was to understand the role of serum TSH in PTC, and in particular if serum TSH can be a progression factor for the tumor. For this purpose, in the present work, we focused on papillary thyroid microcarcinoma. Papillary microcarcinoma, defined as a tumor with size equal to or less than one centimeter in its larger diameter, is today frequently found and its clinical behavior is generally non-aggressive. Increase in number of diagnoses, together with low morbidity and mortality of microcarcinoma raises the problem of its management. Microcarcinoma may be diagnosed as two forms not yet well studied: 1) an “incidental” or “subclinical” microcarcinoma defined as an occult microcarcinoma diagnosed “post-surgery” (occasionally identified on histology in patients submitted to thyroidectomy for other reasons i.e. benign thyroid disease) and 2) “non incidental” or “clinical” microcarcinoma, detected “pre-surgery” because clinically suspected during neck ultrasound or on cytology. Objective of the study is to clarify if these two categories of microcarcinoma reflect the same tumor or are two distinct clinical entities and eventually if serum TSH play a role in their different clinical manifestations .
Patients and Methods: 665 consecutive patients underwent thyroidectomy for benign thyroid disease or for PTC indicative/suspected cytology were recruited. We evaluated preoperative clinical features (thyroid function tests, neck ultrasound, cytology on fine needle aspiration) and histology.
Results: 259 patients presented with a single thyroid nodule and 406 patients with multiple nodules. At histology 92 “incidental” microcarcinomas, 67 “non incidental” microcarcinomas, 215 carcinomas larger than 1 cm and 291 benign nodules were diagnosed.
4 “Incidental” microcarcinoma was significantly more frequent in patients with a multinodular thyroid compared to patients with a single nodule (66/406, 16.2% compared to 26/259, 10%; p=0.02), while in patients with single nodule, “non incidental” microcarcinoma was the most frequent (33/259, 12.7%, compared to 34/406, 8.4%, with multiple nodules, p=0.04). Patients with “incidental” microcarcinoma vs patients with “non incidental” microcarcinoma, were on average older (mean age±standard deviation 53.3±13.2 years compared to 44.9±14.8 in the case of “non incidental” forms; p=0.0002), had a smaller neoplasia (median 4 mm, IQR 2-7 mm, against 9 mm, IQR 7-10 mm of “non incidental” form; Mann-Whitney p<0.0001), more frequently had multiple neoplastic foci (70/92, 76.1 % compared to 35/67, 52.2%; p=0.001) and had lower levels of TSH (median 0.6 mIU /L, IQR: 0.4-1.0 mIU/L, compared to 1.1 mIU/L, IQR: 0.6-1.4 mIU/L; p=0.0001). More aggressive variants were detected more frequently in “non incidental” microcarcinoma (18/67, 26.9% compared to “incidental” microcarcinoma 11/92, 11.9% , χ2 p 0.016), as well as the presence of lymph node metastasis that have been identified in 17/67, 25.4% of patients with “non incidental” form and in any patient with an “incidental” microcarcinoma (χ2 p<0.0001).
Conclusion: “incidental” differs from “non incidental” microcarcinoma in size, clinical diagnosis, multifocality, aggressiveness on histology and in TSH levels: in the presence of lower levels of serum TSH, microcarcinoma is more frequently "subclinical", multifocal, associated with multinodular goiter and has a less aggressive variant on histology. In the presence of higher levels of serum TSH, microcarcinoma is more frequently "clinically” detectable, associated with lymph node metastasis, with a single nodule, less frequently multifocal and has a more aggressive variant. These data indicate that “incidental” and “non incidental” microcarcinomas are two distinct clinical entities with different growth, aggressiveness and eventually prognosis. These features are related to different levels of serum TSH, suggesting a possible effects of this hormone on clinical course of these two cancer presentations.
5 INTRODUCTION
Thyroid nodules
Prevalence
Thyroid nodules are a common clinical problem. Prevalence of palpable nodules is around 5% in women and 1% in men in iodine-sufficient areas [1], with a variability that depends on the area and on iodine availability. Their prevalence increased with the introduction of sensitive techniques such as ultrasound for identification of nodules ranging from 19% to 68% of adult population [1], with a higher frequency in women and in older ages, particularly over 60 years.
Clinical Presentation
Thyroid nodules are more frequently found in the presence of goiter with single or multiple nodules, but can also develop in thyroids with a normal volume. The main problem is to exclude the presence of cancer, which can reach 7-15% of nodules [1] also depending on individual risk factors such as familiarity, ionizing radiation exposure, as well as age and sex.
The introduction of ultrasonography in recent years allowed the identification of clinically occult nodular lesions, with a consequent increase in the incidence of incidental forms [2]. An “incidentaloma” is defined as a thyroid asymptomatic, unexpected nodule, identified during investigations carried out for other conditions and its prevalence, in patients undergoing neck ultrasound, reaches 67% [3]. Clinical prevalence of thyroid nodules, thanks to ultrasound is similar to their prevalence at autopsy [3]. At the same time there has been an increase in detection of nodules smaller than 1 cm as many incidentalomas are small in size and, 70-80% of these, are subcentimeter [3].
These nodules, discovered incidentally have a certain risk of malignancy. This risk ranges from 5-13% if the diagnosis was made by ultrasound, CT or MRI, but as high as 30% if the lesion was identified by the presence of focal uptake on PET with fluorodeoxyglucose [3].
6 In the presence of a thyroid nodule some features at ultrasound may suggest the suspicious of malignancy. This allows a good stratification of the risk of malignancy of the nodular lesion on the basis of its ultrasound pattern and its size and gives indications for the execution of needle aspiration for cytology.
Together with ultrasonogrphy, serum TSH levels add informations on risk of malignancy of thyroid nodules. It has indeed been observed that higher levels of serum TSH, even if in the normal range, are associated with an increased risk of malignancy [4]. Other risk factors of malignancy are family history of thyroid cancer or previous neck irradiation, or a significant increase of nodule size during follow-up.
Ultrasound pattern
Ultrasound study allows to define thyroid gland size and parenchyma features, in particular echogenicity and its homogeneity or heterogeneity. It also helps to identify any nodules and describe their size, location, composition (solid/cystic), echogenicity, margins, presence and kinds of calcifications. Doppler ultrasound study also allows the assessment of the vascularity of the nodule [1].
Use of ultrasound is widely recognized as a key step in risk stratification of malignancy [1] and in decision making for fine-needle aspiration.
The ultrasound pattern of a nodule may be suggestive of malignancy although no sign alone is indicative. The diagnostic value of a combination of several ultrasound features [5], [6], [7], [8], [9], in particular the presence of microcalcifications, a marked hypoechogenicity, irregular margins and tall shape pattern (thickness greater than width) has been studied. Some of these features, even if individually considered, are independent risk factors for malignancy [6]. In a study by Rago et al [5] it has been shown that lack of ultrasound halo was associated with higher probability of malignancy. A combination of several suspicious ultrasound findings had a greater predictive value and high specificity but a low sensitivity [5].
Nodular vascularity includes three kinds of vascular pattern. Nodular blood flow is absent in type I, present and mainly perinodular in tipe II while strongly visible and
7 intranodular in type III. This last kind is one of the striking features of malignancy [6].
Another sign suggesting malignancy is the reduced elasticity of a nodule on elastography [10], [11], [12]. Elastography is a recently introduced technique that allows the evaluation of elastic features of the nodule. It is based on overall ultrasonic beam distortion during application of a pressure by ultrasound probe and depends on the different features of deformability of the hard and soft tissues allowing a semi-quantitative assessment of the elasticity of the nodule [13] . Malignant nodules, in particular papillary thyroid carcinoma, have a lower elasticity than benign lesions [14], [15].
Recently it has been suggested the use of this technique to stratify the risk of cancer in patients with indeterminate or non-diagnostics cytology and to select candidates for surgery [13].
Internal microcalcifications are one of the most specific signs for cancer, in particular for papillary carcinoma [16], but with low sensitivity [6]. Even taller than wider pattern is specific for malignant lesion, reflecting a centrifugal growth of the tumor [16]. In some studies it has been observed an increase in cancer risk according to nodule size, in particular above 2 cm [17], but this finding has not been confirmed in other studies [6], [16].
Presence of a cystic or spongiform pattern, is instead associated with a high probability of benign nodular lesion [16], [17], [18].
In summary, features suggestive of malignancy are: marked hypoechogenicity, infiltrating margins, solid appearance, presence of microcalcifications, reduced elasticity and peri-intranodular vascularity.
Ultrasonography allows a stratification and quantification of risk for malignancy with sensitivity between 87% and 95% and negative predictive value between 88% and 99.8% [3].
By a combination of various sonographic findings, American Thyroid Association guidelines identified five patterns with different risk of cancer [1]:
High suspicion; solid or partially cystic nodule, hypoechoic lesion with one or more of the following features: irregular margins, taller than wide pattern, microcalcifications, marginal calcifications, evidence of extrathyroid extension. The estimated risk of malignancy is 70-90%.
8 Intermediate suspicion; solid hypoechoic nodule with regular margins, without microcalcifications or taller than wide pattern or extension extrathyroid. The risk is around 10-20%.
Low suspicion; iso or hyperechoic nodule, solid or partially cystic lesion with eccentric solid area without microcalcifications or irregular margins, or taller than wide pattern or extrathyroid extension. The risk is around 5-10%.
Very low suspicion; spongiform or partially cystic nodule no suspicious characteristics. The risk is less than 3%.
benign nodule; completely cystic nodule. The risk of malignancy is less than 1%.
Fine needle aspiration
American Thyroid Association guidelines in 2009 [19] recommend the execution of fine needle aspiration for cytological evaluation of solid nodules larger than 1 cm and nodules larger than 5 mm in the presence of ultrasound suspicious findings or individual history of cancer risk.
These guidelines were updated in 2015 [1] on the basis of the introduction of the new risk classification based on ultrasound nodule pattern. Currently fine needle aspiration (FNAB, Fine Needle Aspiration Biopsy) is recommended for nodules:
≥1 cm with high suspicion ultrasound pattern;
≥1 cm with intermediate suspicion ultrasound pattern; ≥1,5 cm with low suspicion ultrasound pattern
Fine needle aspiration may be evaluated for nodules with very low suspicion pattern and ≥2 cm in size, although in these cases follow-up is a reasonable option.
There is no indication for fine-needle for completely cystic nodules.
The diagnostic accuracy of the technique is higher than 90% and the expected number of false negative and false positive is less than 3% [20].
According to series analyzed, 10-40% of nodules submitted to fine needle aspiration presents indeterminate cytology [21] and, of these, 8%-56% revealed malignant histology [22], [23]. To increase sensitivity and specificity of cytological diagnosis use of molecular tests for detection of specific mutations of papillary carcinoma has been proposed.
9 Cytological Classification
Italian consensus for classification and reporting of thyroid cytology [20] identifies the following diagnostic categories:
-THY1 (non-diagnostic lesion); sample is inadequate for presence of artifacts due to fixing or staining or for the presence of blood or for the absence of sufficient cells to make the sample representative The sample is representative when at least 6 groups of 10 well-preserved follicular cells are present.
Inside the THY1 category a subcategory ,THY1C is identified: samples taken from cystic nodules and characterized by the presence of erythrocytes, macrophages full of hemosiderin and cellular debris in abundant colloid, in the absence of sufficient cells to make the sample representative. The authors recommend repeating FNA after at least one month in case of non-diagnostic cytology.
- THY2 (benign lesion). Risk of malignancy is less than 3%. In these cases a clinical and ultrasound follow-ups is recommended and fine needle aspiration repetition if structural modifications or of increase in nodule size.
-THY3 (indeterminate lesion). Samples characterized by the presence of follicular proliferation that may histologically correspond to adenomatous hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma in its follicular variant. We must distinguish two sub-categories: THY3A and THY3B; in the first one neoplastic risk is lower, about 10%, in the second one risk of malignancy reaches 30%. In the first case a afollow-up is suggested, in the second one the first strategy is generally surgery.
- THY4 (suspected malignant lesion). Atypical cells are present and the risk of malignancy is between 60% and 80%.
-THY 5 (malignant lesion). The sample presents cytological features that allow a definitive diagnosis of cancer .
10 Thyroid Cancer
Incidence
Thyroid cancer incidence is about 14.7 new cases per 100,000 inhabitants per year. In recent decades, it has tripled with a more rapid increase since 2000, while mortality is stable around 0.5/100,000[24], [25], [26], [27].
This trend is mainly linked to an increased incidence of papillary thyroid carcinoma (PTC) which increased from 3.4 new cases/100,000 inhabitants per year to 13.1 new cases/100,000 inhabitants, while a lower contribution from follicular histology. According to tumor size, increase of incidence is mainly linked to a more frequent diagnosis of tumors of small dimensions (≤ 2 cm) especially (in the last twenty years) less than 1 cm. Thyroid cancer prevalence increased over 65 years, but, in last decades, its increase affected all age groups although it remains an infrequent cancer under 20 years.
Davies and colleagues [24] developed two hypotheses to explain the increased incidence of papillary carcinoma: a greater identification of subclinical disease or a real increase in incidence. The first one is supported by different studies [29], [30],
[31]. Recent data [33] suggest that increased incidence of thyroid cancer is almost exclusively due to an increase in diagnosis of incidental papillary carcinomas of small dimensions and clinically indolent. This phenomenon of over-diagnosis is probably due to the introduction and the spread of ultrasound and to a greater access to services by the population. The latter phenomenon, in particular, has been observed especially in countries such as Japan and South Korea.
Epidemiological data of Korea, for example, showed that increase in incidence of papillary carcinoma in women was greater in age group between 50 and 59 years in which the highest rate of participation in prevention projects was recorded. Comparing these data with the incidence expected, calculated from incidence curves of the years before the introduction of ultrasound, it has been estimated that 70-80% of cases of thyroid cancer diagnosed in women in Italy, France and the US between 2003 and 2007 it is due to an over-diagnosis phenomenon that would affect a total of 450,000 thyroid cancers in women and 90,000 in men in twelve countries in the last twenty years. On the other hand there is no evidence of
11 changes in exposure to known risk factors or the introduction of new risk factors to justify the important increase incidence rate of thyroid cancer and, in particular, papillary carcinoma [33].
Risk Factors
In the presence of a thyroid nodule, known risk factors for cancer are: age, particularly> 65 years [24], male sex, family history of thyroid cancer and previous neck irradiation in childhood [34]. Exposure to ionizing radiation does not seem to increase the risk of cancer if it occurs in subjects older than 20 years [35]. Nodule fixity with respect to adjacent structures, the presence of symptoms suggestive of extrathyroid extension or laterocervical lymphadenopathy are other
risk factors.
Another important predictor of malignancy is serum TSH [4], [36]. Risk of malignancy increases with the increased levels of TSH, even within the normal range, and higher TSH values are associated with a greater probability of malignancy and with most advanced cancer stage. On the other side treatment with levothyroxine (LT 4), reducing serum TSH, reduces risk of developing thyroid cancer [4]. The risk of PTC is also lower in patients with goiter due to the higher prevalence of functional autonomy and consequently lower TSH levels [36].
Other hypothesized risk factors are diabetes mellitus [37], [38], [39], obesity [40], [41], iodine intake [42], [43], [44], nitrates [45], [46], and Hashimoto's thyroiditis [47], but conflicting data are now available in the literature.
Histological Classification
Neoplasms arising from follicular cells are the majority of thyroid cancers (90%) including:
well differentiated carcinomas that account for about 95% of all follicular tumors. --poorly differentiated or anaplastic cancers , represent 5%.
The first group includes papillary carcinoma (80%), follicular carcinoma (10-15%) and their variants. The main variants of papillary carcinoma are classic and
12 follicular one, more aggressive forms are tall cell variant and diffuse sclerosing one.
Molecular features
Papillary carcinoma shows peculiar molecular abnormalities that may involve several proto-oncogenes. These alterations include mutations affecting BRAF, RAS and RET translocations resulting in a constitutively active gene products able to stimulate cell growth and proliferation. These mutations are mutually exclusive and affect elements belonging to the same route of signal transduction. In recent years, particularly since 2000, an increase in the proportion of RAS point mutations was observed, linked to an increased incidence of follicular variant of papillary carcinoma [24]. Prevalence of BRAF mutations remained largely stable [24] and there has been a reduction in the frequency of RET / PTC translocations, the mutation more related to the exposure to ionizing radiation [48].
RET / PTC
The RET / PTC translocations plays an important role in the genesis of about 30% of papillary carcinomas and is the most common mutation in pediatric tumors and in those associated with ionizing radiation exposure [49]. Several genes involved in RET rearrangement lead to the expression of a chimeric constitutively active receptor which is probably involved in the first steps of tumorigenesis. In support of this hypothesis is a high prevalence of RET translocations in papillary carcinomas of small size. Besides the exposure of cell lines to ionizing radiation leads to the expression of RET / PTC rearrangement[50].
13 B-RAF
Raf is a serine-threonine kinase present in three isoforms (A-Raf, B-Raf and C-Raf) that activates the MAPK transduction pathway of the signal after its activation by RAS. Isoform B o B-RAF is the main activator of MAPK. BRAF mutation is the most frequent genetic alterations in papillary thyroid carcinoma and has been reported in microscopic lesions. This suggests a role in the early stages of tumorigenesis. In addition, BRAF mutation is associated with a higher clinical aggressiveness, risk of extrathyroid extension and a more advanced stage disease [50].
RAS
RAS is a protein with GTPase activity. Three isoforms (NRAS, KRAS, HRAS) are known. RAS is involved in MAPK and PI3K-AKT-mTOR transduction way. RAS mutation is the second most frequent mutation in thyroid carcinoma and the first in follicular carcinoma, but is also present in about 20% of papillary carcinomas [51]. However, its clinical implications and its prognostic value remains unclear [51]. Some studies suggest that some subgroups of patients with RAS mutations may be at risk of dedifferentiation, distant metastasis and reduced survival [52].
14 Papillary microcarcinoma
Definition and prevalence
Papillary thyroid microcarcinoma (microPTC) is defined by the WHO (World Health Organization) as a papillary carcinoma smaller than or equal to 1 cm in its largest diameter [53].
Increased incidence of papillary carcinoma in recent years is mainly due to an increased detection of small tumors [24], about 39% of all papillary carcinomas [27].
Microcarcinoma prevalence at autopsy, in subjects who died from other causes, changes in different countries [54] and can reach 35%, with no difference of distribution in both sexes [55] and with greater prevalence with age [56].
Prevalence of microcarcinoma occasionally seen on neck ultrasound is equal to autopsy prevalence [57].
Clinical p resentatio n
Two different clinical presentation of microcarcinoma are present:
1) an “incidental” or “subclinical” microcarcinoma defined as an occult microcarcinoma diagnosed “post-surgery” (occasionally identified on histology in patients submitted to thyroidectomy for other reasons i.e. benign thyroid disease) 2) “non incidental” or “clinical” microcarcinoma, detected “pre-surgery” because clinically suspected during neck ultrasound or on cytology [57], [58].
If these two kinds of presentation reflect two different clinical entities it is still debated.
Some studies [58], [59], [60] highlight different evolutivity of clinically diagnosed microcarcinoma compared to subclinical one. “Non incidental” microcarcinoma has a greater tendency to progression, may be clinically evident for presence of lymph node localization and has an increased risk of loco-regional recurrence, while “incidental” microcarcinoma has a very low rate of recurrence and substantially absent mortality. These considerations could have an impact in the management and treatment of these tumors.
15 Molecular features
Several studies suggest that no peculiar mutation are present.in microcarcinoma In about 50% of microcarcinomas RET / PTC rearrangement has been identified [61], while BRAF mutations have been identified 17.5%-52% of cases in different studies [62]. These findings suggest a role of such mutations in a early stages of PTC.
In a study of Pontecorvi and coworkers [63] a subgroup of papillary microcarcinomas with a greater clinical aggressiveness and presence of BRAF mutation has been identified. BRAF V600E mutation could be used as a prognostic marker and microcarcinoma aggressiveness [63], [64].
Clinical impact and therapy
Treatment of papillary thyroid microcarcinoma is surgical therapy or active surveillance. Both options allow the same results but surgery is associated with risk of temporary and, more rarely, permanent complications [54], [65]. Considering the low risk of relapse and the very low mortality of microcarcinoma, it appears reasonable a conservative approach [65], although this is not always accepted by the patient for the psychological impact of the disease. In one study, Ito and colleagues [66] demonstrated the possibility of an optimal cancer control in selected patients (who had no lymph node or distance metastasis, with cancer not adjacent to the trachea or to the recurrent laryngeal nerve) through active surveillance. In a period of 10 years, 27.5% of patients showed a growth of more than 2 mm and only 1.2% developed lymph node metastases, thus requiring surgery. However, even in cases where surgery was performed later, oncological control remained excellent and comparable to that obtained with immediate surgery [66].
Based on these considerations it is important to identify patients at low risk that can benefit from a conservative approach.
16 TSH role
TSH action
TSH is the main factor involved in thyrocytes proliferation. TSH binding to its membrane receptor, induces a modulation of gene expression in follicular cell through the activation of two pathways of signal transduction, mediated by a cAMP and by inositol 1,4,5-triphosphate and diacylglycerol. This cause an increased iodine uptake and organification, hormone production and secretion and a stimulus to cell proliferation. The results of studies in animal model suggest that TSH stimulation is involved in the development of follicular cell hyperplasia and cancer. A study conducted on murine model [67], in knock in BRAFV600E mice, showed that mutated BRAF thyrocytes undergo neoplastic transformation process with a short latency period and to an alteration of regulation of genes involved in iodine transport and in thyroid hormones synthesis with development of hypothyroidism. This result in an increase of TSH levels thus promoting proliferation of transformed cells. This observation is in agreement with the finding that, in papillary BRAF mutated carcinomas in humans, decreased expression of thyroid peroxidase, thyroglobulin and iodine transporter (Nis, Na / I symporter) has been found [68]. The importance of TSH in the development of papillary carcinoma was evaluated in BRAF mutated and knock out for TSH receptor mice [67]. It was observed that these animals developed a tumor with attenuated phenotype, characterized by a low mitotic index and by a lower aggressiveness. These results support the important role of proliferative stimulus played by TSH to determine progression and clinical features of neoplastic lesions.
The same result was not obtained with levothyroxine suppressive therapy began shortly after birth in knock in BRAF mice but wild type for TSH receptor [67]. In this model, despite the suppression of TSH, mice developed papillary tumors with the same features of aggressiveness observed in BRAF mutated mice not subjected to levothyroxine therapy. These results seem to suggest that TSH has an
17 important role in the early stages of cancer development. This observation is supported in literature[69].
Clinical relevance
In clinical settings, several studies reported a correlation between risk of malignancy and TSH levels in patients with thyroid nodules. In particular, cancer risk increases with increasing TSH levels even in the normal reference limits. A higher TSH is also more frequent in tumors of greater size and more advanced stage and correlates with the risk of extrathyroid extension [4], [70], [71]. Other authors, however, suggest the existence of a correlation between TSH levels and stage of disease but did not show differences according to nodule size. Instead, they reported differences in TSH values in the presence of extrathyroid extension and lymph node location but a statistical significance was not achieved [72]. Zafon and his collaborators [73] evaluated preoperative TSH levels in patients subjected to surgery for thyroid nodules. They were divided into three groups on the basis of histology (benign nodules, papillary microcarcinoma and larger papillary carcinoma). It was shown that the average TSH values increase gradually in the three groups, but a statistically significant difference was found only between benign disease and papillary macrocarcinoma group. It has also been observed that the presence of thyroid autonomy in patients with goiter, resulting in a reduction of TSH, is associated with a reduced risk of papillary carcinoma [36]. Similarly, levothyroxine therapy, reducing TSH levels, is associated with a reduced incidence of papillary carcinoma. This was observed in a study of Fiore and colleagues [74] in which TSH values and the prevalence of neoplastic lesions were analyzed in two groups of patients with thyroid nodules. In levothyroxine treated group TSH was significantly lower compared to control and this was associated with a prevalence of papillary carcinoma lower than the untreated group.
18 TSH and micro carcino ma
A relationship between papillary microcarcinoma and TSH, however, remains uncertain and several studies report conflicting results in this regard. In a study of Gerschpacher and collaborators [75] TSH levels were not significantly different between patients subjected to thyroidectomy for papillary microcarcinoma and control group consisting of patients undergoing surgery for medullary carcinoma of the thyroid or C-cell hyperplasia. Sohn and coworkers [76] suggested a role of TSH in predicting risk of malignancy in patients with thyroid micronodules. Patients with microcarcinoma showed serum levels of TSH higher than patients with benign micronodules, suggesting that TSH is at least partially involved, possibly with other factors, in the development and / or progression of papillary carcinoma.
Negro and coworkers [77] evaluated a possible role of TSH in the initiation of cancer by comparing preoperative TSH values in two groups of patients undergoing thyroidectomy who received an histological diagnosis of benign nodules or incidental papillary microcarcinoma. In this study no significant differences between the two groups were found, suggesting a role of TSH in the progression of the tumor, rather than in its initiation. Sugitani et al. [78] evaluated a role of TSH as a predictive factor of progression in papillary microcarcinoma. A series of patients with “non incidental” microcarcinoma was followed up for 5 years and both the initial and average values of TSH were considered to assess the presence of a correlation with tumor growth. In this study no significant differences in TSH levels were noted between patients showing stable disease and patients with increased tumor size. According to these data TSH is not a good predictor of progression in patients with microcarcinoma.
Recent data [79] identify TSH as an independent risk factors for the presence of lymph node metastases in patients with microcarcinoma. These results have been confirmed by other studies [80].
19 Overall, reported results in literature are conflicting, probably beacause these studies do not separately evaluate TSH role in “incidental” and “non incidental” forms of microcarcinoma.
20 STUDY
Aim of the study
Aim of this project is to compare clinical and histological features of the two kind of microcarcinoma (clinical and occult microcarcinoma), as well as to evaluate serum TSH levels in the two groups in order to clarify a possible role of serum TSH in different cancer presentation.
Patients and methods
Study design
In this prospective study we evaluated clinical presentation, histology and serum levels of TSH in patients undergoing thyroidectomy.
Patients
We included 665 consecutive patients undergoing thyroid surgery between March 2013 and March 2014. Indications for thyroidectomy were: nodular goiter with compressive symptoms or indeterminate cytology or PTC suspicious/indicative cytology . Of these patients, 202 were men and 463 women and the average age was 50.1±13.8 years. 259 patients presenting with single nodule and 406 with multiple nodules.
21 Inclusion criteria
We included in the study only patients with available clinical data about prior therapies and possible use of drugs affecting serum TSH levels. All patients were subjected to the same diagnostic procedures before surgery. Patients included in the study are summarized in table 1.
Exclusion criteria
One of the aims of the study is to evaluate TSH role in papillary microcarcinoma. For this reason patients receiving levothyroxine, methimazole or medication affecting TSH levels, such as corticosteroids, were excluded . We also excluded patients with Graves' disease, with non papillary thyroid cancer (eg, follicular carcinoma, anaplastic lymphoma), and patients with high levels of calcitonin for medullary carcinoma suspicion.
Diagnostic procedures
Before surgery all subjets were submitted to : -Thyroid ultrasound;
-Tc-99m-pertechnetate scintigraphy in patients with serum TSH below 0.4 mIU/L; -FNAB of dominant cold nodules in patients with goiter, of individual cold nodules larger or equal to 1 cm and of nodules less than 1 cm in the presence of suspicious ultrasound features (for example microcalcifications, irregular margins and marked hypoechogenicity ).
-measurement of TSH, FT3, FT4, thyroid peroxidase and thyroglobulin autoantibodies (TPOAb and TgAb) and calcitonin.
At ultrasound, thyroid volume and presence of nodules were evaluated. Thyroid volume was calculated according to the formula of the ellipsoid model: width x length x thickness x 0.52 for each lobe. A thyroid volume greater than 20 mL in men and 15 ml in women was considered as goiter [81].
22 Patients were divided on the basis of clinical and ultrasound data; patients with multiple nodules were 406 and 335 of these presented goiter while 71 had no goiter.
Patients with single thyroid nodule were 259 and, of these, 122 had a thyroid of normal size while 137 had goiter.
Indication for thyroidectomy
All patients underwent total thyroidectomy. Dissection of neck lymph nodes was performed only in patients with suspicious lymph nodes at ultrasound before surgery.
Of 335 patients with goiter, 141 were subject to thyroidectomy for the presence of compressive symptoms, 143 for the presence of one or more nodules with indeterminate cytology (Thy 3), 51 for PTC suspected or indicative cytology (Thy 4/5).
In the group of patients with multiple nodules, in the absence of goiter, indication for surgery was the presence of Thy 3 (41 patients) or Thy 4/5 (in 29 patients) cytology. One patient underwent to surgery for a clinically suspected nodule, with non-diagnostic cytology (Thy 1), increased in size during follow up.
Of 137 patients, with single nodule and goiter, 31 were subjected to surgery for the presence of large nodules with compression symptoms, 77 for nodules with Thy 3 cytology and 29 for Thy 4/5 cytology.
In the group of patients with single nodule and normal size thyroid 72 patients had a Thy 3 cytology and 50 a Thy 4/5 cytology.
23 Thyroid function tests
Serum tetraiodothyronine (FT4) and free triiodothyronine (FT3) were measured by immunoassay in chemiluminescence (VITROS 3600, Siemens, Buckinghamshire, UK); FT4 normal range: 0.7 to 1.7 ng/dl, FT3 normal range: 2.7 to 5.7 pg/ml.
TSH levels were measured by immunoassay in chemiluminescence (IMMULITE 2000 Third Generation, DPC 5700 Los Angeles, USA), values 0.4-3.4 mIU/L were considered normal.
TgAb and TPOAb were measured by enzyme immunoassay (AIA-Pack Tosoh, Tokyo, Japan). Normal values: TgAb <30 IU/ml, TPOAb <10 IU/ml
Calcitonin was determined by immunoassay in chemiluminescence (IMMULITE 2000, Siemens Healthcare, Llanberis, Gwynedd LL55 4EL, UK), normal values <10 pg/ml.
Fine needle aspiration and cytology
Fine needle aspiration was performed under ultrasound guidance with a needle of 23 gauge 10 ml syringe. Thyroid material was stained with Papanicolaou and Giemsa. Cytological examination results were classified according to the “Italian Consensus for classification and reporting of thyroid cytology” [20].
Histological examination
Surgical specimens were described in their macroscopic features (weight, shape, color and cut surface) and sampled for histological examination by two independent pathologists. All the circumference of the nodules was sampled and samples were obtained for each centimeter of extranodular parenchyma. Thyroid tissue samples were fixed in formalin, embedded in paraffin and stained with hematoxylin and eosin. The histological diagnosis was made in accordance with WHO guidelines [53].
24 Statistic Analysis
Non-parametric variables were described by absolute and relative frequency while quantitative data have been defined through the median and interquartile range (interquartile range, IQR).
Distribution of continuous quantitative variables was assessed by the Kolmogorov-Smirnov test. For quantitative data the Mann-Whitney two-tailed test and the Kruskal-Wallis test were used and for the qualitative variables the Chi-Square test. Differences were considered significant at p <0.05. Statistical analysis was performed by JMP 10 software.
25 Results
Study Group
Patients were divided into four groups according to ultrasound pattern, clinical data, cytology and histology,
1. Patients with papillary “incidental” microcarcinoma (n=92) identified on histology.
In this group, patients were subjected to surgery for multinodular goiters with compressive symptoms and/or single or multiple nodules with indeterminate cytology and a definitive histologic diagnosis of benign nodules. In these patients a microcarcinoma in. parenchyma extranodular was found.
2. Patients with papillary “non incidental” microcarcinoma (n=67), diagnosed before surgery in thyroid with small nodules, occasionally identified at ultrasound and subjected to needle aspiration for the presence of suspicious signs at ultrasound.
3. Patients with papillary carcinoma larger than 1 cm (papillary macrocarcinoma, n =215).
4. Patients with benign nodules (n=291).
“Incidental” microcarcinoma was significantly more frequent in patients with multinodular glands compared to patients with single nodule. It was found in 66 of 406 patients (16.2%) with multiple nodules and in the extranodular parenchyma of 26 of 259 patients (10%, p=0.02) with a single nodule.
On the other hand “non incidental” microcarcinoma was more frequent in patients with a single nodule; it was identified in 33 of 259 patients with single nodule (12.7%) and in 34 of 406 with multiple nodules (8.4%, p = 0.04).
Differences in age distribution were found: patients with “incidental” microcarcinoma were significantly older than those with “non incidental” microcarcinoma with an average age and a standard deviation of 53.3±13.2 years, and 44. 9±14.8 years respectively (p = 0.0002).
Age was not however significantly different between patients with “incidental” microcarcinoma and patients with benign nodules (51.8±12.7 years), nor between
26 patients with “non incidental” microcarcinoma and cancer of more than a centimeter in size (46.7±14.8 years).
There were no sex differences although women were prevalent in all groups. There were no statistically significant difference in frequency of positivity for thyroid autoantibodies in 4 groups. Patients classification according to ultrasound pattern and histology is summarized in table 3.
Tumor size and lymph node metastasis
“Incidental” microcarcinoma was significantly smaller, with a median diameter of 4 mm (IQR 2-7 mm) than “non incidental” one (9 mm with IQR 7-10 mm, Mann Whitney p <0.0001). Median size of macrocarcinoma was 19 mm (IQR 15-30 mm). Size distribution of “incidental” and “non incidental” microcarcinoma is summarized in figure 1.
Lymphadenectomy was performed only in patients with ultrasound suspicious lymph nodes. Lymph node metastases were found at histology in 17/67 (25.4%) patients with “non incidental” microcarcinoma and in no patients with “incidental” microcarcinoma (χ2 p <0.0001). Frequency of lymph node localization in patients with “non incidental” microcarcinoma did not significantly differ from that of patients with carcinoma of more than a centimeter in size (42/215, 19.5%, p = ns).
Histological variants
A total of 374 patients had micro or macrocarcinoma. Of these 285 (76.2%) had less aggressive variants, in particular classical variant was found in 119 cases and follicular variant in 166. More aggressive variants were found in 89 patients (23.7%): in 32 cases a tall cell variant, in 5 a solid and 52 a mixed variant was found. Table 4 shows frequency of different variants in different groups of patients. It was observed that more aggressive variants had a higher frequency in “non incidental” microcarcinoma (18/67, 26.9%) than “incidental” one (11/92, 11.9%; χ2
27 p=0.016), while no statistically significant differences were found between “non incidental” microcarcinoma and macrocarcinoma (60/215, 27.9%).
Multifocality
Presence of multiple foci of cancer was found in 192 cases (51.3% of patients with the tumor).
Multifocality, as shown in the figure 2, was more frequent in “incidental” microcarcinoma than the other two groups. Indeed it was found in 70 cases of 92 “incidental” forms (76.1%), and in 35 of 67 “non incidental” microcarcinomas (52.2%, p=0.001) and in 87 of 215 macrocarcinomas (40.5%, p<0.0001). No significant differences between “non incidental” microcarcinoma and macrocarcinoma were found.
In addition, frequency of multiple neoplastic foci was higher in patients with multiple nodules, being present in 121 of 212 patients with multinodular thyroid (57.1%) compared to 71 of 162 patients with single nodule (43.8%, p =0.01).
No significant differences between patients with multiple nodules with and without goiter (91/163, 55.8% vs. 30/49, 61.2%) or between patients with single nodule with and without goiter (34/72, 47.2% vs. 37/90, 41.1%) were found.
TSH
All patients had normal thyroid function with FT3 and FT4 values in the reference range. TSH median value of the whole group was 0.8 mIU/L with IQR 0.5-1.2 mIU/L. As shown in figure 3, TSH was significantly higher in patients with “non incidental” microcarcinoma compared to “incidental” microcarcinoma (1.1 mIU/L, IQR 0.6-1.4 mIU/L vs 0.6 mIU/L, IQR 0.4-1.0 mIU/L, Mann Whitney p=0.0001) and in the group with macrocarcinoma rather than benign nodules (0.9 mIU/L, IQR 0.6-1.4 mIU/L vs 0.7 mIU/L, IR 0.3 to 1.1 mIU/L, Mann Whitney p<0.0001). No significant differences in TSH levels were observed between patients with
28 “incidental” microcarcinoma and benign nodules or between patients with “non incidental” microcarcinoma and macrocarcinoma
Patients were then divided according to the median TSH value (0.8 mIU/L).
As shown in figure 4, “incidental” microcarcinoma was significantly more frequent than “non incidental” one among patients with TSH ≤0,8 mIU/L (58/92, 63.0% vs 22/67, 32.8%, p <0,0001). Of 92 patients with “incidental” microcarcinoma, 58 had TSH below the median value while only 22 of 67 patients with “non incidental” form (63% compared to 32.8%, p <0.0001).
In patients with TSH> 0.8 mIU/L, “non incidental” microcarcinoma was more frequent than “incidental” one: 45 of 67 patients with “non incidental” microcarcinoma had TSH values greater than 0.8 mIU/L compared to 34 of 92 with “incidental” microcarcinoma (67.1% vs 36.9%, p <0,0001).
TSH levels were also lower in patients with multiple nodules than patients with single nodule (median 0.6 mIU/L, IQR 0.3 to 1.1 mIU/L compared to 0.9 mIU/L, IQR 0,6 to 1.5 mIU/L, Mann Whitney, p <0.0001) as a result of the development of functional autonomy in the first group.
“Incidental” microcarcinoma was more frequent in the group of patients with multinodular gland that had a lower TSH, while “non incidental” microcarcinoma most often appeared as single nodule associated with higher TSH levels.
29 DISCUSSION
In recent years it has been observed an increased incidence of papillary thyroid carcinoma, in particular of small tumors [24]. It is most likely not a real increase in incidence, but is mainly related to subclinical forms detected because of an increased diagnostic sensitivity due to introduction and widespread use of ultrasound and fine-needle method [32], [33], and to different thyroid nodule management [24].
However, clinical benefit of the diagnosis of small tumors, which have very good prognosis and low mortality, must be balanced with the risks of treatment [57], [65], [66].
Risk of recurrence and death from microcarcinoma is, respectively, 3.3% and 0.2% [57]. Many risk factors for recurrence, such as extrathyroid extension, lymph node and distant metastasis are primary related to tumor size and therefore represent a low risk in microcarcinoma [82]. In a study of Oda and colleagues [65] two treatment strategies of microcarcinoma, observation and surgery, were
compared and both had comparable results. In the observation group it was reported a significant increase in lesion size only in 2.3% of patients and the occurrence of lymph node metastases only in 0.5% of the subjects.
In some studies a higher rate of multifocal, local invasion and lymph node metastasis was found in “non-incidental” microcarcinoma compared to “incidental” one and factors allowing the stratification of the risk of recurrence have been investigated [83]. In other studies no evidence of clinical differences between the two forms were found [84]. It remains unclear whether “incidental” and “non incidental” microcarcinoma reflect the same disease or are distinct entities with different own pathogenesis [60].
In our study we observed clinical and histological differences between “incidental” and “non incidental” microcarcinoma.
The first is more frequent in older subjects with goiter. “Non incidental” microcarcinoma has the same features of papillary carcinoma of larger
30 dimensions, was found more frequently in younger patients, presented as a single nodule, was on average larger (9 mm, IQR 7-10 than 4 mm, IQR 2-7 mm; Mann-Whitney p <0.0001) and associated with a higher frequency of lymph node metastases compared to “incidental” one (25% versus 0% in “incidental” forms). More aggressive histological types were more frequent in “non incidental” microcarcinoma (18/67, 26.9%), compared to “incidental” one (11/92, 11.9%; χ2 p = 0.016).
In patients with nodular thyroid disease, the risk of papillary carcinoma increases with increasing levels of serum TSH [4], [80]. TSH levels were significantly higher in patients with macrocarcinoma compared to patients with benign nodules [73]. This finding was also confirmed in our study, in which patients with macrocarcinoma had significantly higher TSH levels than patients with benign nodular disease (0.9 mIU/L, IQR 0.6-1.4 mIU/L vs 0.7 mIU/L, IQR 0.3-1.1 mIU/L, Mann-Whitney p<0.0001). However conflicting data in literature, regarding a relationship between TSH and microcarcinoma are now available [73], [75], [76] perhaps because published studies do not differentiate between “incidental” and clinically diagnosed microcarcinoma.
One aim of this study was to evaluate the relationship between the two kind of presentation of microcarcinoma and TSH levels. TSH was significantly lower in “incidental” microcarcinoma compared to “non incidental” one (0.6 mIU/L, IQR 0.4-1.0 mIU/L compared to 1.1 mIU/L, IQR 0.6 to 1.4 mIU/L). No significant differences in TSH values were found in patients with “incidental” microcarcinoma and benign nodular diseases or in patients with “non incidental” microcarcinoma and macrocarcinoma.
In patients with TSH> 0.8 mIU/L (median value), “non incidental” microcarcinoma was significantly more prevalent than “incidental” one. In patients with TSH< 0.8 mIU/L, “incidental” microcarcinoma was more frequent.
This observation supports the hypothesis of a role of TSH in the presentation and the progression of the disease. TSH is the main factor involved in control of thyrocytes proliferation and is an important growth factor in differentiated thyroid carcinomas. Even in the normal range, TSH, promoting a proliferative stimulus on neoplastic foci, may account for the clinical manifestation of “non incidental” microcarcinoma and could determine its aggressiveness and evolutivity, making it more similar to macrocarcinoma.
31 In “incidental” microcarcinoma more frequently serum TSH is at lower limits of normal range and this may prevent progression of neoplastic lesion. Besides, “incidental” microcarcinoma is more frequently found in patients with goiter that, in its natural history, progressively becomes able to secrete thyroid hormones independently of TSH regulation. This condition is known as functional autonomy and is characterized by reduced serum TSH levels and normal levels of thyroid hormones. Functional autonomy, reducing TSH levels, could slow down papillary cancer development.
An increased frequency of multifocality in “incidental” compared to “non incidental” microcarcinoma (76.1% compared to 52.2%, p=0.001) was also observed. These findings disagree with literature data that support a higher prevalence of multiple foci in “non incidental” microcarcinoma. However, a more recent study by Y.K.So [85] cannot confirm these results, reporting a higher frequency of multifocality in “incidental” form.
One hypothesis that could explain this phenomenon takes into account the pathogenesis of goiter which is also the clinical context in which most frequently “incidental” microcarcinoma occurs. Genetic and environmental factors (figure 5), that contribute to the genesis of goiter, may favor the development of somatic mutations in follicular cells and the initiation of the neoplastic process with formation of multiple foci of papillary carcinoma. In this context, TSH stimulation would be determinant . Serum TSH tends to increase in iodine deficiency and can favor the development of somatic mutations, increasing, on the one hand, proliferative activity of thyrocytes and, second, stimulating their functional activity: thyroid hormones synthesis is associated with hydrogen peroxide production required for iodide anion oxidation. Stimulation of hormone synthesis is then associated with an increased production of reactive oxygen intermediates (ROS = Reactive Oxygen Species) with increased risk of DNA damage and induction of neoplastic process, which may take place in multiple foci. In this context, even activating mutations of TSH receptor or α subunit of the Gs protein, can occur . These mutations are common in goiter and are responsible for the development of functional autonomy of the nodule. Functional autonomy, reducing TSH levels, may slow down cancer progression of multiple foci of papillary carcinoma, preventing its development in a clinically evident cancer.
32 In single nodule, instead, (figure 6) the probability of the initiation of a neoplastic process is lower, but, because of the lack of functional autonomy, higher TSH levels are present, even if normal, stimulating the progression of papillary carcinomas of small dimensions. These “non incidental” forms show the same features of macrocarcinomaand may depend on the presence of TSH stimulation. Even the most frequently aggressive histological variants in “non incidental” microcarcinoma, along with TSH, may allow a faster growing, a larger size and a greater frequency of lymph node metastases compared to “incidental” forms.
33 CONCLUSIONS
“Incidental” and “non incidental” microcarcinoma show different clinical and histological features. “Non incidental” microcarcinoma has the same behavior of larger tumors and is on average larger compared to “incidental” one, may be diagnosed through lymph node metastasis, most often is in a single nodules and histologically aggressive variants are more common. “Incidental” microcarcinoma is on average smaller, more often diagnosed in patients with multiple nodules, no lymph node metastases have been detected and more frequently multiple tumor foci in the extranodular parenchyma are identified. The presence of multiple nodules and patient's age are associated with an increased likelihood of development of functional autonomy in multinodular goiter, that causes a
progressive reduction of serum TSH.
TSH levels are significantly different in the two forms of microcarcinoma, supporting the role of this hormone in tumor initiation and progression: clinically detectable forms have normal levels of TSH while, in “incidental” forms, TSH is significantly lower. In the first case, higher levels of TSH promote the progression of the lesion making it clinically detectable, in the second case the development of functional autonomy, through inhibition of TSH secretion, reduces neoplastic foci stimulation preventing its clinical presentation.
34 REFERENCES
[1] B. R. Haugen, E. K. Alexander, K. C. Bible, G. Doherty, S. J. Mandel, Y. E. Nikiforov, F. Pacini, G. Randolph, A. Sawka, M. Schlumberger, K. G. Schuff, S. I. Sherman, J. A. Sosa, D. Steward, R. M. Tuttle and L. Wartofsky, “2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer,” Thyroid, vol. 26, no. 1, p. thy.2015.0020, 2015.
[2] D. S. Ross, “Editorial: Nonpalpable thyroid nodules - Managing an epidemic,” J. Clin. Endocrinol. Metab., vol. 87, no. 5, pp. 1938–1940, 2002. [3] G. Russ, S. Leboulleux, L. Leenhardt, and L. Heged??s, “Thyroid
incidentalomas: Epidemiology, risk stratification with ultrasound and workup,” Eur. Thyroid J., vol. 3, no. 3, pp. 154–163, 2014.
[4] E. Fiore and P. Vitti, “Serum TSH and risk of papillary thyroid cancer in nodular thyroid disease,” J. Clin. Endocrinol. Metab., vol. 97, no. 4, pp. 1134–1145, 2012.
[5] T. Rago, P. Vitti, L. Chiovato, S. Mazzeo, A. De Liperi, P. Miccoli, P. Viacava, F. Bogazzi, E. Martino, and A. Pinchera, “Role of conventional ultrasonography and color flow-doppler sonography in predicting malignancy in ‘cold’ thyroid nodules,” Eur. J. Endocrinol., vol. 138, no. 1, pp. 41–46, 1998.
[6] E. Papini, R. Guglielmi, A. Bianchini, A. Crescenzi, S. Taccogna, F. Nardi, C. Panunzi, R. Rinaldi, V. Toscano, and C. M. Pacella, “Risk of Malignancy in Nonpalpable Thyroid Nodules : Predictive Value of Ultrasound and Color-Doppler Features,” vol. 87, no. June 1995, pp. 1941–1946, 2016.
[7] E. Kim, C. S. Park, K. K. Oh, D. I. Kim, and J. T. Lee, “Aspiration Biopsy of Nonpalpable Solid Nodules of the Thyroid,” no. March, pp. 687–691, 2002. [8] C. Cappelli, M. Castellano, I. Pirola, E. Gandossi, E. De Martino, D. Cumetti,
B. Agosti, and E. A. Rosei, “Thyroid nodule shape suggests malignancy,” Eur. J. Endocrinol., vol. 155, no. 1, pp. 27–31, 2006.
35 [9] W.-J. Moon, S. L. Jung, J. H. Lee, D. G. Na, J.-H. Baek, Y. H. Lee, J. Kim, H. S. Kim, J. S. Byun, and D. H. Lee, “Benign and malignant thyroid nodules: US differentiation--multicenter retrospective study.,” Radiology, vol. 247, no. 3, pp. 762–770, 2008.
[10] P. Trimboli, R. Guglielmi, S. Monti, I. Misischi, F. Graziano, N. Nasrollah, S. Amendola, S. N. Morgante, M. G. Deiana, S. Valabrega, V. Toscano, and E. Papini, “Ultrasound sensitivity for thyroid malignancy is increased by real-time elastography: A prospective multicenter study,” J. Clin. Endocrinol. Metab., vol. 97, no. 12, pp. 4524–4530, 2012.
[11] J. Bojunga, N. Dauth, C. Berner, G. Meyer, K. Holzer, L. Voelkl, E. Herrmann, H. Schroeter, S. Zeuzem, and M. Friedrich-Rust, “Acoustic Radiation Force Impulse Imaging for Differentiation of Thyroid Nodules,” PLoS One, vol. 7, no. 8, pp. 1–8, 2012.
[12] Y. F. Zhang, H. X. Xu, Y. He, C. Liu, L. H. Guo, L. N. Liu, and J. M. Xu, “Virtual Touch Tissue Quantification of Acoustic Radiation Force Impulse: A New Ultrasound Elastic Imaging in the Diagnosis of Thyroid Nodules,” PLoS One, vol. 7, no. 11, pp. 1–8, 2012.
[13] T. Rago, M. Scutari, F. Santini, V. Loiacono, P. Piaggi, G. Di Coscio, F. Basolo, P. Berti, A. Pinchera, and P. Vitti, “Real-time elastosonography: Useful tool for refining the presurgical diagnosis in thyroid nodules with indeterminate or nondiagnostic cytology,” J. Clin. Endocrinol. Metab., vol. 95, no. 12, pp. 5274–5280, 2010.
[14] T. Rago, F. Santini, M. Scutari, A. Pinchera, and P. Vitti, “Elastography: New developments in ultrasound for predicting malignancy in thyroid nodules,” J. Clin. Endocrinol. Metab., vol. 92, no. 8, pp. 2917–2922, 2007.
[15] Y. Hong, X. Liu, Z. Li, X. Zhang, M. Chen, and Z. Luo, “Real-time Ultrasound Elastography in the Differential Diagnosis of Benign and Malignant Thyroid Nodules,” J. Ultrasound Med., vol. 28, no. 7, pp. 861–867, 2009.
[16] J. P. Brito, M. R. Gionfriddo, A. Al Nofal, K. R. Boehmer, A. L. Leppin, C. Reading, M. Callstrom, T. A. Elraiyah, L. J. Prokop, M. N. Stan, M. H. Murad, J. C. Morris, and V. M. Montori, “The Accuracy of Thyroid Nodule Ultrasound to Predict Thyroid Cancer: Systematic Review and Meta-Analysis,” J. Clin. Endocrinol. Metab., vol. 99, no. 4, pp. 1253–1263, Apr. 2014.
36 [17] R. B. Goldstein, N. Brasic, and C. Jin, “Characteristics : Results of a
Population-Based Study,” vol. 173, no. 19, pp. 1788–1796, 2014.
[18] J. A. Bonavita, J. Mayo, J. Babb, G. Bennett, T. Oweity, M. Macari, and J. Yee, “Pattern recognition of benign nodules at ultrasound of the thyroid: Which nodules can be left alone?,” Am. J. Roentgenol., vol. 193, no. 1, pp. 207–213, 2009.
[19] American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, D. S. Cooper, G. M. Doherty, B. R. Haugen, B. R. Hauger, R. T. Kloos, S. L. Lee, S. J. Mandel, E. L. Mazzaferri, B. McIver, F. Pacini, M. Schlumberger, S. I. Sherman, D. L. Steward, and R. M. Tuttle, “Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer,” Thyroid Off. J. Am. Thyroid Assoc., vol. 19, no. 11, pp. 1167–1214, 2009.
[20] F. Nardi, F. Basolo, A. Crescenzi, G. Fadda, A. Frasoldati, F. Orlandi, L. Palombini, E. Papini, M. Zini, A. Pontecorvi, and P. Vitti, “Italian consensus for the classification and reporting of thyroid cytology,” J. Endocrinol. Invest., vol. 37, no. 6, pp. 593–599, 2014.
[21] Y. E. Nikiforov, D. L. Steward, T. M. Robinson-Smith, B. R. Haugen, J. P. Klopper, Z. Zhu, J. a Fagin, M. Falciglia, K. Weber, and M. N. Nikiforova, “Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules.,” J. Clin. Endocrinol. Metab., vol. 94, no. 6, pp. 2092–2098, 2009.
[22] L. Yassa, E. S. Cibas, C. B. Benson, M. C. Frates, P. M. Doubilet, A. A. Gawande, F. D. Moore, B. W. Kim, V. Nosé, E. Marqusee, P. R. Larsen, and E. K. Alexander, “Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation,” Cancer, vol. 111, no. 6, pp. 508–516, 2007.
[23] Z. W. Baloch, S. Fleisher, V. A. LiVolsi, and P. K. Gupta, “Diagnosis of ‘follicular neoplasm’: A gray zone in thyroid fine-needle aspiration cytology,” Diagn. Cytopathol., vol. 26, no. 1, pp. 41–44, Jan. 2002.
[24] L. Davies, L. G. T. Morris, M. Haymart, A. Y. Chen, D. Goldenberg, J. Morris, J. B. Ogilvie, D. J. Terris, J. Netterville, R. J. Wong, G. Randolph, and on behalf of the A. E. S. S. Committee, “AMERICAN ASSOCIATION
37 OF CLINICAL ENDOCRINOLOGISTS AND AMERICAN COLLEGE OF ENDOCRINOLOGY DISEASE STATE CLINICAL REVIEW: THE INCREASING INCIDENCE OF THYROID CANCER,” Endocr. Pract., vol. 21, no. 6, pp. 686–696, Jun. 2015.
[25] L. Davies and H. Welch, “INcreasing incidence of thyroid cancer in the united states, 1973-2002,” JAMA, vol. 295, no. 18, pp. 2164–2167, May 2006.
[26] A. Y. Chen, A. Jemal, and E. M. Ward, “Increasing incidence of differentiated thyroid cancer in the United States, 1988-2005,” Cancer, vol. 115, no. 16, pp. 3801–3807, 2009.
[27] L. Davies and H. Welch, “Current thyroid cancer trends in the united states,” JAMA Otolaryngol. Neck Surg., vol. 140, no. 4, pp. 317–322, Apr. 2014. [28] D. T. Hughes, M. R. Haymart, B. S. Miller, P. G. Gauger, and G. M. Doherty,
“The Most Commonly Occurring Papillary Thyroid Cancer in the United States Is Now a Microcarcinoma in a Patient Older than 45 Years,” Thyroid, vol. 21, no. 3, pp. 231–236, Jan. 2011.
[29] H. R. Harach, K. O. Franssila, and V. M. Wasenius, “Occult papillary carcinoma of the thyroid. A ‘normal’ finding in Finland. A systematic autopsy study.,” Cancer, vol. 56, no. 3, pp. 531–538, 1985.
[30] F. J. Martinez-Tello, R. Martinez-Cabruja, J. Fernandez-Martin, C. Lasso-Oria, and C. Ballestin-Carcavilla, “Occult carcinoma of the thyroid. A systematic autopsy study from Spain of two series performed with two different methods.,” Cancer, vol. 71, no. 12, pp. 4022–9, 1993.
[31] J. D. MORTENSEN, L. B. WOOLNER, and W. A. BENNETT, “GROSS AND MICROSCOPIC FINDINGS IN CLINICALLY NORMAL THYROID GLANDS,” J. Clin. Endocrinol. Metab., vol. 15, no. 10, pp. 1270–1280, Oct. 1955.
[32] J. P. Zevallos, C. M. Hartman, J. R. Kramer, E. M. Sturgis, and E. Y. Chiao, “Increased thyroid cancer incidence corresponds to increased use of thyroid ultrasound and fine-needle aspiration: A Study of the veterans affairs health care system,” Cancer, vol. 121, no. 5, pp. 741–746, 2015.
[33] S. Vaccarella, S. Franceschi, F. Bray, C. P. Wild, M. Plummer, and L. Dal Maso, “Worldwide Thyroid-Cancer Epidemic? The Increasing Impact of Overdiagnosis,” N. Engl. J. Med., vol. 375, no. 7, pp. 614–617, Aug. 2016. [34] S. J. Schonfeld, C. Lee, and A. Berrington de González, “Medical Exposure
38 to Radiation and Thyroid Cancer,” Clin. Oncol., vol. 23, no. 4, pp. 244–250, Jul. 2016.
[35] K. Furukawa, D. Preston, S. Funamoto, S. Yonehara, M. Ito, S. Tokuoka, H. Sugiyama, M. Soda, K. Ozasa, and K. Mabuchi, “Long-term trend of thyroid cancer risk among Japanese atomic-bomb survivors: 60 years after exposure,” Int. J. Cancer, vol. 132, no. 5, pp. 1222–1226, 2013.
[36] E. Fiore, T. Rago, M. A. Provenzale, M. Scutari, C. Ugolini, F. Basolo, G. Di Coscio, P. Berti, L. Grasso, R. Elisei, A. Pinchera, and P. Vitti, “Lower levels of TSH are associated with a lower risk of papillary thyroid cancer in patients with thyroid nodular disease: Thyroid autonomy may play a protective role,” Endocr. Relat. Cancer, vol. 16, no. 4, pp. 1251–1260, 2009.
[37] D. Schmid, G. Behrens, C. Jochem, M. Keimling, and M. Leitzmann, “Physical activity, diabetes, and risk of thyroid cancer: a systematic review and meta-analysis,” Eur. J. Epidemiol., vol. 28, no. 12, pp. 945–958, 2013. [38] S.-R. Shih, W.-Y. Chiu, T.-C. Chang, and C.-H. Tseng, “Diabetes and thyroid
cancer risk: literature review.,” Exp. Diabetes Res., vol. 2012, no. Table 1, p. 578285, 2012.
[39] C.-H. Tseng, “Thyroid cancer risk is not increased in diabetic patients.,” PLoS One, vol. 7, no. 12, p. e53096, 2012.
[40] C. M. Kitahara, E. A. Platz, L. E. B. Freeman, A. W. Hsing, S. Linet, Y. Park, C. Schairer, A. Schatzkin, J. M. Shikany, and A. B. De González, “NIH Public Access,” Biomarkers, pp. 1–15, 2012.
[41] Z. G. Zhao, X. G. Guo, C. X. Ba, W. Wang, Y. Y. Yang, J. Wang, and H. Y. Cao, “Overweight, obesity and thyroid cancer risk: a meta-analysis of cohort studies,” J Int Med Res, vol. 40, no. 6, pp. 2041–2050, 2012.
[42] É. Cléro, F. Doyon, V. Chungue, F. Rachédi, J.-L. Boissin, J. Sebbag, L. Shan, F. Bost-Bezeaud, P. Petitdidier, É. Dewailly, C. Rubino, and F. de Vathaire, “Dietary Iodine and Thyroid Cancer Risk in French Polynesia: A Case–Control Study,” Thyroid, vol. 22, no. 4, pp. 422–429, Jan. 2012.
[43] U. Feldt-Rasmussen, “Iodine and Cancer,” Thyroid, vol. 11, no. 5, pp. 483– 486, May 2001.
[44] M. Knobel and G. Medeiros-Neto, “Relevance of iodine intake as a reputed predisposing factor for thyroid cancer.,” Arq. Bras. Endocrinol. Metabol., vol. 51, no. 5, pp. 701–712, 2007.
